The present invention relates to transgene expression systems, related compositions, including pharmaceutical compositions, and methods of making and using them. Preferred systems employ an adenovirus transgene expression vector comprising a DNA molecule (transgene) having a sequence encoding a desired product, expressibly contained within an adenovirus vector containing at least a portion of the E3 region and certain portions of the E4 region. The E4 portions comprise the open reading frame sequence known as E4ORF3 and at least one other portion of E4. Preferably the E4 portion of the vector (or xe2x80x9cE4 cassettexe2x80x9d) includes E4ORF3 and at least one other portion selected from E4ORF4, E4ORF6/7 and E4ORF3/4. The invention has a number of important features including improving persistency of transgene expression in a desired host cell. The transgene expression systems of the present invention are useful for a variety of applications including providing persistent cellular expression of the transgene in vitro and in vivo.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb. The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. See generally, Horwitz, M. S., xe2x80x9cAdenoviridae and Their Replication,xe2x80x9d in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990.
Recombinant adenoviruses have advantages for use as transgene expression systems, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (see e.g., Berkner, K. L., Curr. Top. Micro, Immunol., 158:39-66 (1992); Jolly D., Cancer Gene Therapy, 1:51-64 (1994)).
Adenovirus vectors can accommodate a variety of transgenes of different sizes. For example, an approximately eight (8) kb insert can be accommodated by deleting regions of the adenovirus genome dispensable for growth (e.g., E3). Development of cell lines that supply nondispensable adenovirus gene products in trans (e.g., E1, E2a, E4) has allowed insertion of a variety of transgenes throughout the adenovirus genome (see e.g. Graham, F. L., J. Gen. Virol., 36:59-72 (1977); Imler et al., Gene Therapy, 3:75-84 (1996)). For example, the p53, dystrophin, erythropoietin, ornithine transcarbamylase, adenosine deaminase, interleukin-2, alantitrypsin, thrombopoietin, and cytosine deaminase genes have all been individually inserted into the adenovirus genome for making expression vectors.
The natural tropism of adenoviruses for respiratory tract cells has made them attractive vectors for the treatment of cystic fibrosis (CF): the most common autosomal recessive disease in Caucasians. In CF, mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene disturbs cAMP-regulated Cl-channel function, resulting in pulmonary dysfunction. The CFTR gene has been introduced into adenovirus vectors to treat CF in several animal models and human patients. Particularly, studies have shown that adenovirus vectors are fully capable of delivering CFTR to nasal epithelia of CF patients, as well as the airway epithelia of cotton rats and primates. See e.g., Zabner et al., Nature Genetics, 6:75-83 (1994); Rich et al., Human Gene Therapy, 4:461-476 (1993); Zabner et al., Cell, 75:207-216 (1993); Zabner et al., Nature Genetics, 6:75-83 (1994); Crystal et al., Nature Genetics, 8:42-51 (1994); Rich et al., Human Gene Therapy, 4:461-476 (1993).
Importantly, recent studies have demonstrated that it is possible to restore a functioning chloride ion channel in CF patients by providing an adenoviral vector encodinge CFTR to airway epithelia cells (Zabner et al., J. Clin. Invest., 97:1504-1511 (1996)).
However, in vitro and in vivo studies have pointed to opportunities to further improve such vectors. For example, transgene expression from adenovirus vectors is often transient. Persistent transgene expression is highly desirable in gene therapy settings, especially those seeking to alleviate chronic or hereditary disease in mammals. At least some of the limitations are due to induction of a cell-mediated immune response against infected cells. In particular, cytotoxic: T lymphocytes (CTLs) have been detected against antigenically expressed viral proteins encoded by adenovirus vectors, even though such vectors are replication defective. CTLs have also been detected against immunogenic transgene products. Cytotoxic T lymphocytes have the potential destroy or damage cells harboring the adenovirus vectors, thereby causing loss of transgene expression. Cell destruction can also cause inflamation which is also detrimental to the tissues involved. The cell-mediated immune response can pose a potentially serious obstacle to therapies requiring high dosages, which are likely to elicit more potent immune responses. See J. Kaplan et al., Human Gene Therapy 8:45-56 (1997); Y. Yang et al., Proc. Nat. Acad. Sci. 91:4405-11(1994); Y. Yang et al., J. Virol. 70:7202 (1996).
Various strategies have been used to minimize cell-mediated immune responses induced by adenovirus vectors. Generally, the strategies include modulation of the host immune response itself or engineering adenovirus vectors with a decreased capacity to induce immune responses.
For example, co-administration of immunosuppressive agents and adenovirus vectors have been reported to prolong persistence of transgene expression (Fang et al., Hum. Gene Ther., 6:1039-1044 (1995); Kay et al., Nature Genetics, 11: 191-197 (1995); Zsellenger et al., Hum. Gene Ther., 6:457-467 (1995)).
In another approach, modification of adenovirus genome sequences in recombinant vectors has been used in attempts to decrease recognition of the vector by the immune system (see e.g., Yang et al., Nature Genetics, 7:362-369 (1994); Lieber et al., J. Virol., 70:8944-89600 (1996); Gorziglia et al., J. Virol., 70:4173-4178 (1996); Kochanek et al., Proc. Natl. Acad. Sci. USA, 93:5731-5736 (1996); Fisher et al., Virology, 217:11-22 (1996)).
The choice of promoter or transgene may also influence persistence of transgene expression from adenovirus vectors (see e.g., Guo et al., Gene Therapy, 3:801-802 (1996); Tripathy et al., Nature Med., 2:545-550 (1996)).
Persistence of transgene expression from adenovirus vectors has been reported to be influenced by the adenovirus E3 gp19K protein. That protein can complex with M.HC Class I molecules in the-endoplasmic reticulum, thereby preventing both cell surface presentation of viral antigens and killing of transduced cells by cytotoxic, T-lymphocytes (CTLs) (Wold et al., Trends Microbiol., pp. 437-443, (1994)). However, approaches based on that knowledge have only achieved limited success.
Another problem which has faced researchers who are attempting to utilize viral vectors for gene therapy has been the size of the heterologous DNA which can be inserted into the modified viral genome. Early work involving insertion of heterologous genes in the area of E1 deletion resulted in vectors which were difficult to produce in sufficient quantities to permit continued clinical testina. See, e.g., D. Armentano et al., Hum. Gene Ther., 6:1343 at 1344 (1995). While it is possible to possible to produce viral vectors which contain adenoviral DNA that is longer than the wild-type genome length, the ability to replicate such vectors can decrease precipitously when the wild-type genome length is substantially exceeded.
Accordingly, there is a need to develop and produce transgene expression vectors that have a genome whose size permits packaging that provides persistent transgene expression and minimizes cell-mediated immune reactions against cells containing the vectors. Such vectors would have a variety of uses in delivering genes to cells, including use as gene transfer vectors in gene therapy.
The present invention relates to transgene expression systems and related compositions; including pharmaceutical compositions, and methods of making and using same. For example, in one aspect, the invention relates to an adenovirus transgene expression vector comprising a heterologous DNA molecule (transgene) contained within an adenovirus vector in which the adenovirus vector contains at least a portion of the E3 region, and a modified E4 region. The modified E4 region may include only E4ORF4, or preferably, the modified E4 region contains at least E4ORF3 and at least one other portion of the E4 region. The term xe2x80x9cmodified EV as used herein refers to an E4 region which has at least one deletion. Preferably, the size of the deletion(s) is (are) sufficient, taking into consideration any deletions elsewhere in the viral genome and also any added DNA, such as the heterologous DNA (transgene(s)) and associated promoters, enhancers and other transcriptional control elements, that the size of the resulting viral vector is typically less than about 110%, more preferably less than about 105%, most preferably less than about 101% of the size of the wild-type virus genome. Preferably the deletion comprises at least one E4 open reading frame, although deletions in non-ORF portions of the E4 region can also be utilized. As indicated above, some modified E4 regions of the vectors of the present invention require expression from E4ORF3, and at least one additional portion of the E4 region. More preferably the E4 deletion comprises a plurality of open reading frames. Optionally, E4ORF6 may be deleted.
For example, a deletion includes removal of an ORF by frameshift mutation or knock-out which does not effect an open reading frame of another ORF.
In another aspect, the invention relates to an adenovirus transgene expression vector comprising DNA sequence encoding a transgene contained within an adenovirus vector in which the adenovirus vector contains at least a portion of the E3 region, and E4ORF4, or a modified E4 region which contains at least E4ORF4.
Preferably the modified E4 portion included in the vector of the present invention (or xe2x80x9cE4 cassettexe2x80x9d) includes E4ORF3 and at least one other portion selected from E4ORF4, E4ORF6/7 and E4ORF3/4. Preferred parts of the E4 cassette are E4ORF3 and E4ORF4, E4ORF3 and E4ORF6/7, and E4ORF3 and E4ORF3/4. The modified E4 portion may also comprise E4ORF6. Thus far, the most preferred combination for persistent transgene expression from mammalian cells transformed with the vectors of the present invention is the combination of E3 with E4ORF3 and E4ORF4. The transgene expression system facilitates persistent expression of the transgene in cells and protects the cells from cell-mediated immune responses. The invention also features pharmaceutical compositions that comprise the transgene expression system and methods for using of such compositions to deliver therapeutic transgenes to desired cells. The invention further features methods for the production of the transgene expression system, as well as methods for using the transgene expression stem to express a transgene in cells, tissues and mammals, and methods of protecting cells expressing transgenes from cell-mediated immune responses.
A transgene expression system that facilitates persistent expression of transgenes in cells or groups of cells (e.g., tissue) has been developed. In general, the transgene expression system persistently expresses a desired transgene in the presence of expression of at least a portion of the adenovirus E3 region (xe2x80x9cE3 cassettexe2x80x9d) and a modified E4 region, preferably E4ORF3 and at least one other portion of E4. The transgene expression system achieves persistent transgene expression and protects cells expressing the transgene from cell-mediated immune responses, thereby providing more stable transgene expression in the cells. Accordingly, the present transgene expression system has a variety of uses, including use as a gene transfer system for providing desired transgenes to cells in vitro and in vivo.
Thus, in one aspect of the invention, transgene expression systems are provided that comprise (a) DNA molecule encoding a transcription unit comprising a transgene operably linked to expression control sequences, (b) at least a portion of adenovirus E3) region, and (c) E4ORF3 and least one other portion of E4. In this aspect, the transgene expression system facilitates persistent expression of the transgene in cells and protects the cells expressing the transgene from cell-mediated immune responses that can potentially damage or destroy the cells harboring the transgene expression system (i.e., transduced cells). Accordingly, use of the transgene expression system improves transgene expression, e.g., by prolonging survival of cells expressing the transgene, and by reducing inflammation caused by the destruction of such cells.
In general, the transgene expression systems of the present invention provide significant advantages. For example, use of prior transgene expression systems typically stimulates host immune responses (e.g., CTLs, inflammation) that damage or destroy transduced cells expressing a desired transgene. In contrast, the present transgene expression systems provide persistent transgene expression while minimizing loss of expression caused by cell-mediated immune responses. Use of the prior transgene expression systems in high dosages typically induce pronounced cell-mediated immune responses against transduced cells. In contrast, the present transgene expression systems minimizes the susceptibility of transduced cells to cell mediated immune (CMI) responses, thereby making the present invention attractive for situations where repeat administration of the transgene may be required, e.g., for use in gene therapy treatments requiring high or multiple dose protocols.
Additionally, prior transgene expression systems using recombinant adenovirus typically required high multiplicities of infection (MOIs) for efficient use. Often, high inputs of the virus killed transduced cells e.g., by introducing cytotoxic amounts of viral protein. In contrast, the present transgene expression system provides the opportunity to achieve persistent transgene expression without relying on undesirable amounts of input virus. Thus, use of the present transgene expression systems improve viability of cells expressing transgenes.
Generally, the transgene expression systems of the present invention provide persistent transgene expression in transduced mammalian cells, due at least in part to the presence of the adenovirus E3 region, at least (but not restricted to) the portion thereof coding for the gp19K gene product of adenovirus E3 region. Persistence can also be improved by inclusion in the vector of those portions of the E3 region which provide one or more of the following: the 14.7k protein, the 10.4k protein, and the 14.5k protein.
Persistance can also be improved by including the E4ORF3 and E4ORF4 or other portion of the adenovirus E4 region. The presence of these DNA segments in the vector facilitates persistent expression of the transgene in cells while minimizing or eliminating destruction by the cell-mediated immune responses against cells expressing the transgene.
In preferred embodiments, transgene expression systems of the present invention are provided as replication-defective adenovirus vectors. That is, the replication-defective vectors are unable to propagate on host cells at a level normally achieved by wild-type adenovirus (e.g., Ad2, Ad5) or replication-competent adenovirus-based vectors. Thus, in a preferred embodiment, the DNA sequence encoding the transcription unit, at least a portion of the adenovirus E3 region, E4ORF3, and at least one other portion of E4, preferably E4ORF4, E4ORF6/7 and/or E4ORF3/4, are provided together (i.e. in cis) on a single replication-defective adenovirus vector. A number of replication defective adenovirus vectors are known in the field including vectors"" deficient in E1 region function such as those adenovirus vectors lacking the E1a and E1b regions.
The components of a transgene expression system according to the invention can be configured on a single replication-defective adenovirus vector in a number of ways. For example, as reported in published PCT application WO 96/30534 (herein incorporated by reference), it is possible to construct a variety of adenovirus vectors in which a transcription unit comprising a transgene (i.e., expression cassette) is positioned in the E1a and E1b region of the adenovirus vector and an E4 region cassette is positioned in the E4 region. However, in other adenovirus vectors, the expression cassette is positioned in the E4 region and the E4 cassette is positioned in the E1a, E1b regions.
Thus, in accordance with the invention, the transcription unit comprising the transgene is constructed and inserted into an adenovirus vector in accordance with conventional recombinant DNA methods. A particularly preferred insertion site is the E1a/E1b region of the adenovirus vector. In that same vector, the adenovirus E3 region portion and the E4 cassette are preferably positioned in the E3 and E4 regions of the adenovirus vector, respectively.
Alternatively, the transcription unit comprising the transgene may be inserted in the E4 region of the adenovirus vector and the E4 cassette can advantageously be inserted in the E1a/E1b region of the adenovirus vector. In such a case, the E3 cassette is preferably positioned, e.g., in the E3 region of the adenovirus vector.
In a preferred embodiment of the invention, the transcription unit comprising the transgene is inserted in an E1a/E1b region of an adenovirus vector. In this embodiment, the transcription unit and the adenovirus E3 region portion are provided on a single adenovirus vector, separated by adenovirus genome sequences comprising the E2 region. The adenovirus vector is further provided with at least a portion of the adenovirus E3 region, E4ORF3, and at least one other portion of E4, preferably E4ORF4, E4ORF6/7 and/or E4ORF3/4, positioned distally to the adenovirus E3 region of the vector relative to the direction of adenovirus major late transcription portions. The adenovirus vector is capable of providing persistent transgene expression and protecting transduced cells from damaging or destructive cell-mediated immune response by expressing the transgene in the presence of expression from at least a portion of the adenovirus E3 region and at least E4ORF3 and E4ORF4 and/or other portions of the adenovirus E4 region.
Expression of the transgene is augmented by an operably linked eukaryotic or viral transcriptional control elements such as promoters, enhancers, and other control elements suitable for use with adenovirus vectors. Preferably, a strong promoter is linked 5xe2x80x2 to the transgene so as to drive transgene expression in a variety of cell types. For example, a strong promoter particularly suited for use with the E4ORF3 gene product (e.g., the cytomegalovirus (CMV) or polyglycerol kinase (PGK) promoters may be included so as to enhance transgene expression. In such embodiments, the transgene also includes DNA encoding one or more RNA processing signals, preferably a polyadenylation segment.
Additionally, in preferred embodiments. the transcription unit further comprises sequence modifications that reduce production of replication-competent adenovirus during laboratory propagation of the adenovirus vector and further provide for making clinical-grade preparations thereof (see e.g., Guo et al. supra).
The E3 cassette in the adenovirus vector may comprise a full length E3 region. Alternatively, the E3 cassette may constitute a portion of the E3 region, preferably a portion capable of adversely effecting operation of major histocompatibility class I (MHC class I) molecules in cells expressing the transgene expression system. For example, the gp19K gene product of the E3 region, has been shown to down-regulate MHC presentation. A reduction or elimination of MHC presentation in cells containing the transgene expression systems of the invention augments the persistence of the transgene by reducing or eliminating susceptibility to cell-mediated immune responses against such cells. Examples of E3 cassettes comprising the gp19K region have been disclosed in a co-pending U.S. patent application Ser. No. 08/839,553, now U.S. Pat. No. 6,020,191, entitled xe2x80x9cNovel Adenovirus Vectors Capable of Facilitating Increased Persistence of Transgene Expressionxe2x80x9d filed on Apr. 14, 1997, the disclosure of which is incorporated herein by reference. See also copending application U.S. patent Ser. No. 08/839,552, now U.S. Pat. No. 5,981,275, entitled xe2x80x9cNovel Transgene Expression Systems For Increased Persistencexe2x80x9d filed Apr. 14, 1997, the disclosure of which is incorporated by reference. In addition, the 14.7, 10.4 and 14.5k proteins of E3 may also be included to provide additional protection against lysis by TNF-xcex1 (see Wold et al., Virology 184:1-8 (1991)).
As noted, in addition to the E3 region or portion thereof. the vector contains E4ORF3 and at least one other portion of E4, preferably E4ORF4, E4ORF6/7 and/or E4ORF3/4. Preferred parts of the E4 cassette are E4ORF3 and E4ORF4, E4ORF3 and E4ORF6/7, and E4ORF3/4. The most preferred embodiment includes E3, E4ORF3, and E4ORF4. The next most preferred embodiment includes E3, E4ORF3, and E4ORF6/7.
The transgene expression systems of the present invention can be configured to contain more than one DNA construct. For example, the DNA sequence encoding the transcription unit, at least a portion of the adenovirus E3 region, the E4ORF3 segment or portion thereof, and the E4ORF4 and/or other portion of the adenovirus E4 region may preferably be provided in the same DNA construct, e.g., a replication deficient adenovirus vector. In such embodiments, a user of the present invention can readily handle and introduce the components of the transgene expression system simultaneously into cells. Thus it is preferred that the DNA sequence encoding the transcription unit, and the required regions of the adenovirus be included together on a single adenovirus vector.
However, it is also an object of the present invention to provide the components of the transgene expression system on separate DNA constructs, e.g., plasmids, adenovirus vectors, recombinant adenovirus derived from the adenovirus vectors and combinations thereof, thereby giving the user control over administration of the transgene expression system. Particularly, this approach is advantageous in several settings, e.g., in instances where it is desirable to introduce components of the transgene expression system to cells at different times.
For example, the DNA sequence encoding the transcription unit may alternatively be included on a single plasmid, and the adenovirus E3 and E4 cassettes may be included in a separate adenovirus vector. In other embodiments, the DNA sequence encoding the transcription unit and the E3 cassette region is included in a plasmid and the E4 cassette is included in a separate adenovirus vector. Alternatively, the DNA sequence encoding the transcription unit and the adenovirus E4 cassette may be included in the plasmid, and the adenovirus; E3 region may be included in the adenovirus vector. In addition to plasmids, components of the transgene expression system can be provided on a circular DNA capable of autonomous replication e.g., in a bacterial host such as a phagemid, cosmid, episome, or the like.
Additionally, the components of the transgene expression system can be positioned on one or more suitable plasmids, phagemids, cosmids, or episomes.
In one embodiment of the present invention, a transgene expression system described previously is provided as a complex comprising at least one cationic amphiphile to facilitate delivery and entry into of the transgene expression system to target cells. In a preferred embodiment, the transgene expression system is provided as a composition comprising the transgene expression system, preferably as a complex comprising the transgene expression system and at least one cationic amphiphile, in which the composition is capable of delivery and expression of a transgene to a cell to achieve a particular desired effect. For example, the transgene is a functional wild-type CFTR gene delivered to airway epithelial cells and the phenotypic effect is providing a functional chloride ion channel to such cells. Preferably, the composition can alleviate a chronic or hereditary disease, including those disorders or diseases, afflicting mammalian respiratory tissue (e.g., cystic fibrosis).
In other embodiments of the present invention, the composition comprises a transgene expression system comprising at least a portion of the E3 region, the E4ORF3 and at least one other portion of E4, preferably E4ORF4, E4ORF6/7 and/or E4ORF3/4, together with a carrier. The composition may comprise a transgene expression system capable of reducing expression of MHC Class I receptor in cells containing that system, thereby improving longevity of transgene expression. The composition may comprise a transgene expression system in which the E3 region of the transgene expression system comprises at least the DNA sequence encoding gp19K protein. In another embodiment, the composition comprises a transgene expression system in which the E3 region consists of DNA encoding gp19K, 14.7k, 10.4k and 14.5k proteins and the E4 cassette consists of E4ORF3 and E4ORF4. Preferably, the transgene is operably linked to a eukaryotic promoter, such as the CMV or a PGK promoter, more preferably the CMV promoter. The transgene preferably comprises a gene which is capable of being expressed to provide a gene product which can alleviate disease or disorder such as occurring in manimalian respiratory tissue. In preferred embodiments, the composition comprises a transgene expression system in which the transgene is the wild-type cystic fibrosis transmembrane regulator (CFTR) gene.
In accordance with the present invention, there is provided a method of making a composition as described previously, in which the method comprises combining the transgene expression system with one or more cationic amphiphiles sufficient to form the composition. Optionally, the composition further comprises a carrier.
The invention is also directed to methods for the production of the transgene expression system, as further described below.
The present invention further provides methods for expressing a transgene in cells, tissues or mammals and reducing or eliminating susceptibility to cell-mediated immune responses against cells expressing the transgene in which the method comprises combining a transgene expression system of the invention claim with one or more suitable cationic amphiphiles to form a complex and administering the complex to cells in the mammal or contacting the cells with the complex under conditions sufficient to transform the cells and express the transgene in the cells to achieve the desired phenotype, and reducing or eliminating susceptibility to the cell-mediated immune response by expressing the transgene in the presence of an expressed E3 cassette and a modified E4 cassette in accordance with this invention.